The present invention relates to methods for the separation of particles in a fluidic microsystem, especially under the action of electrophoresis, and to fluidic microsystems set up to perform such methods.
The separation of microobjects such as, e.g., particles with a natural or synthetic origin or molecules in fluidic microsystems under the action of electrically or magnetically induced forces is becoming increasingly more significant in biomedical and chemical analytical technology. Two conventional separating principles that differ basically according to the type of electrical separating forces are schematically illustrated in FIGS. 10A, B.
FIG. 10A schematically shows the separation by means of negative dielectrophoresis (see, e.g., DE 198 59 459). Particles with different dielectric properties flow in a fluidic microsystem 100′ through a first channel 30′. A field barrier extending transversely over channel 30′ is generated with electrode arrangement 40′ by subjecting it to high-frequency electrical fields which barrier is permeable or acts in a laterally deflecting manner in cooperation with the flow forces as a function of the dielectric properties of the particles. Particles 22′ with a permittivity (or conductivity) that is low in comparison to the medium are deflected into adjacent channel 30A′ whereas particles 21′ with a higher permittivity (or conductivity) flow further in channel 30′. Since the dielectrophoresis is a function of the particle size (see T. Schnelle et al. in “Naturwissenschaften”, vol. 83, 1996, pp. 172-176), a separation of the particles in accordance with their size can take place even given the same dielectric properties. The conventional dielectrophoretic particle separation can have disadvantages as concerns the reliability of the separation, in particular in the case of particles with similar permittivities, and as concerns the complexity of the channel design. The reliability of the separation can be limited, in particular in the separation of biological cells of the same type into different subtypes (e.g., macrophages, T lymphocytes, B lymphocytes).
Another problem that has been solved only in a limited fashion in the conventional dielectrophoretic separation of particles can be given by the occurrence of undesired cell components in biological suspension specimens. Cell components can frequently not be distinguished from complete cells solely by their dielectrophoretic properties. Furthermore, they can result in microsystems in undesired accumulations and channel constrictions and in cloggings strong enough to cause system failure. Finally, undesired cell components can also have a disturbing effect on measurements of cells such as, e.g., on a patch-clamp measurement. There is therefore interest in an improved process for purifying suspension specimens that has a greater reliability than the dielectrophoretic separation of particles.
FIG. 10B illustrates an electrophoretic separation of particles, e.g., molecules in a microstructured channel (see T. Pfohl et al. in “Physik Journal”, vol. 2, 2003, pp. 35-40). Electrodes 41′, 42′, are arranged on the ends of channel 30′ formed with alternating broad and narrow sections, which electrodes form an electrophoretic field in channel 30′ when subjected to a direct voltage. The drift rate of the molecules in the electrophoretic field is a function of their molecular weight and charge. In the wider sections of channel 30′ the drift rate of the larger molecules is less, so that in the course of the separation at first the small molecules and later the large molecules arrive at the end of the separation path. The electrophoretic separation in fluidic microsystems does have the advantage that the use of a separation gel as in macroscopic electrophoresis can be eliminated. However, the principle shown in FIG. 10B has the disadvantage that a separate microsystem with adapted geometric parameters must be provided for each separation task and in particular for each particle type. It is also disadvantageous that the separation takes place in the liquid at rest because this is associated with a great amount of time involved and with additional measures for adaptation to continuous systems.
The above-cited separation principles are also mentioned in WO 98/10267. Charged particles are drawn, e.g., electrophoretically from a specimen into a buffer solution flowing in parallel in the channel of a fluidic microsystem. This technique is limited to specimens with certain properties of the specimen components. Furthermore, it is disadvantageous since the particles can be drawn electrophoretically onto the channel walls, which is undesirable, especially in the case of biological material, e.g., cells.
The electrophoretic deflection of particles is also described in DE 41 27 405. Particles are moved in a resting liquid under the action of electrical traveling waves. When they pass electrophoresis electrodes during the movement, a separation takes place in accordance with the electrical properties of the particles. The same disadvantages result as in above-cited WO 98/10267.
The combining of dielectrophoretic and electrophoretic field effects in the manipulation of particles in fluidic microsystems is also known. According to DE 195 00 683 particles suspended in liquid are held in an electrode arrangement that forms a closed field cage (potential well) when loaded with high-frequency alternating voltages by negative dielectrophoresis. In order to correct variations in position caused by thermal conditions, particles in the field cage are additionally shifted electrophoretically. The electrophoretic shifting takes place within the framework of a control circuit in accordance with the positional variations of the particle, that are determined, e.g., optically. The technology described in DE 195 00 683 is not suitable for particle separation since it constitutes a closed, stationary measuring system. Furthermore, the combination of dielectrophoresis and electrophoresis on the closed field cage is limited to relatively large individual particles. Disadvantages can result during the measuring, e.g., of macromolecules since in their case the action of negative dielectrophoresis is distinctly less than that of electrophoresis, so that an undesired accumulation of macromolecules on the electrodes can occur. Particle groups cannot be measured with this technique since all particles require their own correction movement. A separation of particles would also be rendered more difficult by a dipole-dipole effect (see T. Schnelle et al. in “Naturwissenschaften”, vol. 83, 1996, pp. 172-176), which furthers an aggregation of particles.
DE 198 59 459 also teaches the combination of alternating and direct voltages in fluidic microsystems for the targeted fusion or poration of cells. The action of direct voltage on the fusion or poration is limited in this technique and a particle separation is not provided.
The publication of S. Fiedler et al. in “Anal. Chem.”, vol. 67, 1995, pp. 820-828 teaches generating temporary or local pH gradients that can be verified with fluorescent dyes by an optionally pulsed direct voltage control of microelectrodes in aqueous electrolyte solutions.
There is not only an interest in a separation of particle mixtures according to geometric (size, shape) or electrical properties (permittivity, conductivity) for pharmacological, analytical and biotechnological research but also according to other parameters such as, e.g., surface charges or charge-volume ratios. The occurrence of surface charges is described, e.g., by N. Arnold et al. in “J. Phys. Chem.”, vol. 91, 1987, pp. 5093-5098; L. Gorre-Talini et al. in “Phys. Rev. E” vol. 56, 1997, pp. 2025-2034; and Maier et al. in “Biophysical J.” vol. 73, 1997, pp. 1617-1626.
The object of the invention is to provide improved methods for the separation of particles in liquid flows in fluidic microsystems with which the disadvantages of conventional techniques are avoided. Methods in accordance with the invention should be characterized in particular by an expanded area of application for a plurality of different particles and by increased reliability in particle separation. The object of the invention is also to provide improved microsystems for the implementation of such processes, in particular improved microfluidic separating devices characterized by a simplified construction, great reliability, simplified control and a broad area of application for different types of particles.